So, you’ve tricked your favorite E. coli expression strain into making gobs of your protein of interest (POI). The SDS-PAGE gel shows a whopping excess of what you have decided to nickname ‘OCD-Linked Zelda Legendase’. Great! This is always an encouraging result, but now you must determine how best to lance the little living bags of proteinaceous gold so it might permit you to prove your hypothesis, publish and prosper!

Initial lysis should be simple, utilizing a system such as Gold Biotechnology’s Bacterial Cell Lysis Buffer Kit. You will, of course, want to be sure to keep the bacterial cell debris pellets from the centrifugation step for downstream analysis. If your next gel shows that most of the POI has been captured among the soluble proteins in the lysate, huzzah! You can now proceed to the welcome challenge of isolating your POI via size exclusion, ion exchange or affinity chromatography employing, e.g., GoldBio’s High Density Nickel Resin.There is nothing more satisfactory than a single band on the gel, is there?

However, if the protein is relatively insoluble, as evidenced by its localization in the pellet or its tendency to crash out of solution at some point during purification, it may be necessary to take advantage of a denaturing agent such as the nonionic 8M urea or the chaotropic 6M guanidine-HCl (GdnHCl). One can utilize these agents to solubilize protein found in inclusion bodies, which occupy the dubious position of the dreaded `rat kings’ (see image below) of protein purification.

Inclusion bodies abstractly similar to the aforementioned rat king consist of a disordered aggregate of partially denatured recombinant proteins that are associated with each other via principally hydrophobic interactions. Statistics obtained from the Center for Eukaryotic Structure Genomics from more than 8,000 cloned targets expressed in E. coli showed that only 30% of the proteins were expressed in soluble form, with most accumulating to some extent in inclusion bodies. Researchers have traditionally employed a `one-step denaturing and refolding procedure’, which allows refolding of up to 40% of the included protein (1).

A 'two step' method recently superseded the former technology to allow recovery of more than 75% of the inclusion body protein. Instead of using either 6M GdnHCl or 8M urea to denature the protein prior to refolding, the two-step method initially used both denaturants in succession.

In this method, one carries out two cycles of denaturation and refolding while simply switching denaturant. More recently, an improvement on the two step technique has been developed by the Wang lab at Fudan University that utilizes L-Arginine as the initial denaturant in place of GdnHCl (2). Protein recovered using this method appears to very closely mimic the fluorescent properties of recombinant eGFP from the soluble fraction(see eGFP, shown at right).

Expression and purification of a novel protein can prove to be a difficult and demanding quest, even when one has the tremendous advantage of an appropriate affinity resin-packed column fastened under one’s belt. The specter of encountering inclusion bodies looms large over any such journey, but with high-quality reagents such as GoldBio’s urea and uanidine-HCl in hand, one can feel confident that the goal is in reach.

1) Burgess, R. R. (2009). Refolding solubilized inclusion body proteins. Methods in enzymology, 463, 259-282.

2) Yang, Z., Zhang, L., Zhang, Y., Zhang, T., Feng, Y., Lu, X., ... & Wang, X. (2011). Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PloS one, 6(7), e22981.

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